A covalent inhibitor targeting the papain-like protease from SARS-CoV-2 inhibits viral replication

Covalent inhibitors of the papain-like protease (PLpro) from SARS-CoV-2 have great potential as antivirals, but their non-specific reactivity with thiols has limited their development. In this report, we performed an 8000 molecule electrophile screen against PLpro and identified an α-chloro amide fragment, termed compound 1, which inhibited SARS-CoV-2 replication in cells, and also had low non-specific reactivity with thiols. Compound 1 covalently reacts with the active site cysteine of PLpro, and had an IC50 of 18 μM for PLpro inhibition. Compound 1 also had low non-specific reactivity with thiols and reacted with glutathione 1–2 orders of magnitude slower than other commonly used electrophilic warheads. Finally, compound 1 had low toxicity in cells and mice and has a molecular weight of only 247 daltons and consequently has great potential for further optimization. Collectively, these results demonstrate that compound 1 is a promising lead fragment for future PLpro drug discovery campaigns.


SARS CoV-2 infection of cells
Vero 6 cells or Calu 3 cells were seeded in 96-well plates (40,000 cells/well) and allowed to grow in DMEM supplemented with 10% (v/v) FBS, 0.05 mg/mL penicillin G and 80 µg/mL streptomycin and incubated at 37°C with 5% CO2 for 24 h. Cells were treated with compounds for 1 hour, infected with SARS-CoV-2 at a MOI of 0.05 for Vero 6 and 0.1 for Calu 3 for 72 hours. Remdesivir was included as a positive control. A 150 µL of supernatant was collected, fixed with 500 µL Trizol (in deep 96-well plates), and stored at -80°C for RNA extraction to determine viral RNA levels by qRT-PCR. Cells were then washed with PBS and fixed with 4% PFA, fixed cells were permeabilized and stained with a dsRNA antibody to detect viral replication. Figure S1. Levels of intracellular double stranded RNA in Vero E6 cells infected with SARS-CoV-2 in the presence of various concentrations of compound 19 and compound 20. Percent dsRNA was determined by normalizing against DMSO-treated infected cells.

qRT-PCR
100 µL of cell culture fluids were collected and mixed with 300 µL Trizol or RLT buffer. RNA was extracted and purified using the PureLink RNA mini kit (Invitrogen) according to the manufacturer's instructions. RNA was analyzed with a SuperScript One-step qRT-PCR kit to quantify viral RNA copy numbers. The forward primer (ACAGGTACGTTAATAGTTAATAGCGT) and reverse primer (ATATTGCAGCAGTACGCACACA) were used to amplify the SARS-CoV-2 E RNA.

Mouse MTD assay
Ethical statement The animal study was reviewed and approved by the University of California, Berkeley Animal Care and Use Committee(ACUC) and Laboratory Animal Care(OLAC) AUP-2019-04-12046. All the mice were purchased from Jackson laboratory (Maine, USA) 20 mg/kg of compound 1 was administered to 3 female Balb/c mice (6-8 weeks old) via intraperitoneal injection. The mouse body weight was monitored for 7 days. The results are shown in Figure S2.

Molecular Dynamics Simulation of PLpro with compound 1
To model the dynamics of compound 1 inside the PLpro active site, we drew the chemical structure of compound 1 in MarvinSketch. OpanBable was used to convert 2D chemical structures into 3D. The protein structure of SARS-CoV-2 PLpro (PDB: 6W9C) was obtained from RCSB server. The CHARMM36 forcefield was used to simulate protein ligand interactions, the CGENFF server was used to optimize forcefield parameters for compound 1. Molecular Dynamics (MD) simulation of PLpro and compound 1 interactions were performed with GROMACS. In all cases, we put the protein-ligand complex inside the water box with 1 nm spacing from each side, then we neutralized the system with Na + and Cl -. In order to model water molecules dynamics, we used the TIP3P forcefield which considers three sites for each molecule. To insert compound 1 with energy minimized structures inside the active site of PLpro, we used 1-ClickDocking online server, which provides final structures of drugs positioned outside of the active site. After getting noncovalent docking results from 1-ClickDocking server, we used VMD to manually put drugs inside the active side near CYS111. Now we can use these new complex structures to model covalent inhibition of PLpro using MD simulation. We used particle mesh Ewald (PME) method to capture long range electrostatic interactions. To equilibrate the system, we did NVT and NPT simulations for 100 ps with V-rescale thermostat and Berendsen barostat. This general MD setup is similar to prior studies that our group has done. The MD production simulations were set to model the dynamics of the system in 30 ns using V-rescale thermostat and Parrinello-Rahman barostat. For the covalent interaction between PLpro CYS111 and ligands, we manually added a thioether between the sulfur atom of CYS111 and compound 1. Finally, the structures of PLpro combined with compound 1 were visualized with VMD.

LC-MS/MS analysis of PLpro incubated with compound 1
Purified PLpro (20 μg) in 100 μL buffer of 150 mM NaCl, 20 mM HEPES, 5 mM TCEP was incubated for 30 min at room temperature with 10 μM Compound 1. The sample was precipitated by addition of 25 µL of 100% (w/v) trichloroacetic acid before cooling to −80 °C for 1 h. Following incubation, the sample was centrifuged at max speed for 10 min at 4 °C. The supernatant was carefully removed and the sample washed 3X with ice-cold 0.01 M HCl/90% acetone solution. The pellet was then resuspended in 4 M urea containing 0.1% Protease Max (Promega, V2071) and diluted in 40 mM ammonium bicarbonate buffer. 10 mM TCEP was then added and the samples were incubated at 60 °C for 30 min. Next, 12.5mM iodoacetamide was added and the sample incubated for another 30 minutes at 37 °C. The sample was then diluted 50% with PBS before sequencing grade trypsin (1 μg per sample, Promega, V5111) was added for an overnight incubation at 37 °C. The following day, the sample was centrifuged at 13,200 rpm for 30 min, and the supernatant transferred to a new tube and acidified to a final concentration of 5% formic acid and stored at −80 °C until mass spectrometry analysis.

Preparation of alpha-Chloro Amides: General Procedure A:
A 5 mL solution of chloroacetyl chloride (0.69 mmol, 1.1 eq) in dry dichloromethane is dropwise added to a 5 mL solution of amine (0.63 mmol, 1 eq) in dry dichloromethane at room temperature. After 4 hours of stirring, the reaction mixture is filtered over a fritted Buchner funnel to obtain solid product. Preparation of alpha-Chloro Amides: General Procedure B: A 5 mL solution of chloroacetyl chloride (0.69 mmol, 1.1 eq) in dry dichloromethane is dropwise added to a 5 mL solution of amine (0.63 mmol, 1 eq) and 250 uL triethylamine in dry dichloromethane at room temperature. After 4 hours of stirring, the reaction mixture is filtered over a fritted Buchner funnel to obtain solid product. subsequently added to the enzyme solution. The mixed solutions were incubated at room temperature for 20 min. 50 µL of Mpro protease substrate solution was added to each well, followed by 30 min incubation at 37 °C. The Mpro activity was detected by measuring the fluorescence emission intensity λ excitation=490 nm; λ emission=520nm. Figure S5: Compounds 1, 19, and 20 have no interaction with SARS-CoV-2 Mpro(3CL).

Fluorescence based PLpro activity assay in the presence of dithioerythritol (DTT) and glutathione (GSH)
PLpro inhibition assays were performed in the presence of 5 mM DTT and GSH to determine if compound 1 was active in the presence of thiols. PLpro protease activity was measured with peptide substrate x-x-G-G-AMC (Bachem) in the presence or absence of 5 mM DTT and GSH. PLpro was added into a black, flat-bottom 96-well plate at a final concentration of 50 nM with reaction buffer [150 mM NaCl, 20 mM HEPES, 0.1mg/mL BSA, pH 7.5] with or without 5 mM DTT or 5 mM GSH]. Compound 1 was added to the enzyme solution at concentrations ranging from 0-80 µM for 20 min at room temperature. The enzyme activity assay was initiated by mixing the x-x-G-G-AMC probe at a final 50 µM and incubating for another 20 min at room temperature. The reaction was ended by adding 1 µL of aqueous citric acid. The PLpro activity was detected by measuring the fluorescence emission intensity λ excitation=360 nm; λ emission=460nm.